Risk factors for recurrences and visual impairment in patients with ocular toxoplasmosis: A systematic review and meta-analysis

Background Ocular toxoplasmosis (OT) is caused by the parasite Toxoplasma gondii. OT is the leading cause of posterior uveitis globally; it is a recurrent disease that may result in visual impairment and blindness. This systematic review and meta-analysis aim to summarize and evaluate the risk factors for recurrences, visual impairment, and blindness described in the literature worldwide. Methods and findings We performed a systematic literature search in PubMed, Embase, VHL, Cochrane Library, Scopus, and DANS EASY Archive. All studies reporting patients with clinically and serologically confirmed OT presenting any clinical or paraclinical factor influencing recurrences, visual impairment, and blindness were included. Studies presenting secondary data, case reports, and case series were excluded. An initial selection was made by title and abstract, and then the studies were reviewed by full text where the eligible studies were selected. Then, the risk of bias was assessed through validated tools. Data were extracted using a validated extraction format. Qualitative synthesis and quantitative analysis were done. This study was registered on PROSPERO (CRD42022327836). Results Seventy two studies met the inclusion criteria. Fifty-three were summarized in the qualitative synthesis in three sections: clinical and environmental factors, parasite and host factors, and treatment-related factors. Of the 72 articles, 39 were included in the meta-analysis, of which 14 were conducted in South America, 13 in Europe, four in Asia, three multinational, two in North America and Central America, respectively, and only one in Africa. A total of 4,200 patients with OT were analyzed, mean age ranged from 7.3 to 65.1 year of age, with similar distribution by sex. The frequency of recurrences in patients with OT was 49% (95% CI 40%–58%), being more frequent in the South American population than in Europeans. Additionally, visual impairment was presented in 35% (95% CI 25%–48%) and blindness in 20% (95% CI 13%–30%) of eyes, with a similar predominance in South Americans than in Europeans. On the other hand, having lesions near the macula or adjacent to the optic nerve had an OR of 4.83 (95% CI; 2.72–8.59) for blindness, similar to having more than one recurrence that had an OR of 3.18 (95% CI; 1.59–6.38). Finally, the prophylactic therapy with Trimethoprim/Sulfamethoxazole versus the placebo showed a protective factor of 83% during the first year and 87% in the second year after treatment. Conclusion Our Systematic Review showed that clinical factors such as being older than 40 years, patients with de novo OT lesions or with less than one year after the first episode, macular area involvement, lesions greater than 1 disc diameter, congenital toxoplasmosis, and bilateral compromise had more risk of recurrences. Also, environmental and parasite factors such as precipitations, geographical region where the infection is acquired, and more virulent strains confer greater risk of recurrences. Therefore, patients with the above mentioned clinical, environmental, and parasite factors could benefit from using prophylactic therapy.

Introduction Ocular Toxoplasmosis (OT) is caused by the parasite of the apicomplexan family known as Toxoplasma gondii (Tg), considered a neglected tropical disease [1]. Moreover, OT is the leading cause of posterior uveitis worldwide; its prevalence and clinical features varies according to region due to the different host and parasite factors [2][3][4].
Recurrences may occur in 40% to 79% of cases, generating visual impairment and potentially blindness [5][6][7]. The best treatment for OT is controversial and, there is no consensus on which drugs must be used for prophylactic treatment of recurrences [8][9][10][11]. Although, some risk factors for recurrences, such as the number of lesions (patients with only one lesion have a 60% greater risk than those with more than 2 lesions) [12], presence of polymorphisms (the presence of T/A heterozygosis in the IFN-γ gene at position AT+874 had a 49% higher risk of recurrences than those with A/A homozygosis) [12], and age (higher risk of recurrences was associated with older patients that present a de novo active lesion) [12][13][14] have been identified, it is still unclear which patients should receive prophylaxis.
Additionally, some data comes from studies with low-quality evidence, generating weak recommendations regarding risk factors for recurrence and visual outcomes, treatment and prophylactic treatment in OT. Thus, this topic deserves an update and synthesis of the available literature to provide high-quality evidence that leads to strong recommendations. Therefore, this systematic review and meta-analysis aimed to evaluate the factors that increase the risk of recurrence, visual impairment, and blindness in patients with OT.

Methods
This systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (S1 Checklist) [15]. It was registered in the "International prospective register of systematic reviews" (PROSPERO ID: CRD42022327836). Due to the characteristics of our study, it does not need to have ethical committee approval.

Search strategy
We conducted a systematic literature search in the following databases: PubMed, Embase, VHL (Virtual Health Library), Cochrane Library (Ovid), Scopus, and DANS EASY Archive. We used "MeSH," "Emtree, and "DeCS" terms accordingly. The search strategy can be found in S1 Table. We identified and deleted duplicated articles with the assistance of Zotero and Excel filters.

Selection criteria
We reviewed papers that evaluated patients with a diagnosis of OT confirmed by clinical and/ or serological criteria exposed to any clinical or paraclinical factor influencing our outcomes of interest, including recurrences or visual impairment.
Studies eligible for inclusion in this systematic review included clinical trials (randomized/ non-randomized) and observational studies (case-control, cohort, cross-sectional studies, and population-based studies). We excluded secondary studies (systematic, narrative, and scoping reviews), case reports, case series, and articles from preclinical studies (not providing clinical outcome data) and abstracts.

Definitions
Recurrence of OT was defined as the presence of an active retinochoroidal lesion associated with scarring in at least one eye (congenital and acquired) [7]. Additionally, the World Health Organization definitions for visual impairment and blindness were used [16].

Selection process
Nine authors (AO, CC, EC, FV, LM, MS, MH, PB, WR) formed five pairs of review authors how independently examined the titles and abstracts identified by the electronic searches. Each author screened titles and abstracts independently to exclude those that were unrelated to our question based on the selection criteria; then, the independent decision was compared with the pair, and any disagreements were discussed. If disagreements persisted, a panel of seven thematic experts decided if the article should be included or excluded. The level of agreement in each couple was: C1 = 81.1% (LM+PB), C2 = 82.2% (FV+EC), C3 = 72.4% (MS+CC), C4 = 79.4% (MH+WR), and C5 = 96.2% (AO+WR). The overall agreement was 82.4%. The step-by-step can be found in Fig 1.

Data extraction
Data were extracted by nine independent investigators (AO, CC, EC, FV, LM, MS, MH, PB, WR) using a standardized and validated Excel form. The following characteristics were extracted from each eligible study: article code, author, year, digital object identifier system, design of the study, intervention, country, studied population, age, race, sex, immunological status, Tg serotype/genotype, ocular inflammation characteristics, number of reactivations, factors related to the reactivations and magnitude of the risk, frequency of visual impairment and blindness, factors related to the visual impairment and blindness and magnitude of the risk, treatment, duration of treatment, type of prophylactic treatment, duration of prophylactic treatment, and sociodemographic factors. For all included studies, mean ± standard deviation (SD) or median and interquartile range (IQR) were used for data extraction.

Risk of bias assessment and quality assessment
To assess the risk of bias for randomized clinical trials (RCTs), the version 2 of the Cochrane risk-of-bias tool for randomized trials (RoB 2) was used [17]. This tool includes five specific bias domains: randomization; deviation from intended intervention; missing data; outcome

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measurement; and selection of reported results. To evaluate the risk of bias in Non-Randomized studies of interventions, the ROBINS-I tool was used [18], which considered seven domains: confounding; selection of participants; classication of intervention; deviation from interventions; missing outcome data; measurement of outcomes; selection of reported result. The figures were done using the Risk-of-bias VISualization (robvis) [19].
Moreover, quality of cross-sectional studies was assessed by the Agency for Healthcare Research and Quality (AHRQ) tool [20]; the score was categorized as high risk of bias (0 to 4 score), moderate risk of bias (5 to 7 score), and low risk of bias (8 to 11 score) [21]. Finally, to evaluate the risk of bias in longitudinal studies, cohorts, and case-control, the checklist provided by the Clinical Advances Through Research and Knowledge Translation (CLARITY) group of McMaster University was used [22]. Detailed information can be found in S2 Table. Data synthesis and statistical analysis For prevalence, a meta-analysis of forest plots was conducted using the R Package (dmetar version 0.0.9000) [23,24], and for related factors, forest plots Review Manager (RevMan 5.4; The Nordic Cochrane Centre, The Cochrane Collection, Copenhagen, Denmark) were used. A random effects model was used for all analyses, considering the significant heterogeneity of data. Only variables that were reported by at least two included studies underwent metaanalysis.
Since our primary outcome was to evaluate the factors influencing the recurrences rate, visual impairment, and blindness, we initially looked for the frequency of recurrences, visual impairment, and blindness subdivided by continents. The precaution of not including randomized and non-randomized clinical trials was always considered since they do not represent the reality of the world situation as they are controlled studies. Moreover, for the frequency analyses of visual impairment and blindness, we analyzed the final visual acuities reported in longitudinal and cross-sectional studies, and data was managed in eye categories because most studies report the number of eyes instead of patients. Studies that did not provide this data were excluded from the meta-analysis and included in the systematic review.
The influence of the following characteristics in the primary outcomes was evaluated: laterality of the disease, retinal location, sex, number of lesions, complications, and type of treatment. This analysis was done considering the number of patients, not the number of eyes. In order to assess the heterogenicity, we used the I 2 statistics test with 30% to 60% as representing moderate heterogeneity and 50% to 90% representing substantial heterogeneity. Publication bias was evaluated using funnel plots if there were more than 10 studies. Signicance was set at the level of P-value less than 0.05.
Finally, for the qualitative synthesis, we created tables summarizing the most important factors related to recurrences and visual outcomes (visual impairment or blindness). They were subdivided according to the following categories: clinical and environmental factors, parasite and host factors, and therapeutic factors. All articles that provided relevant data for qualitative analysis were summarized, regardless they were included or not in the meta-analysis.

Study selection
We found 1,119 articles, of which 193 duplicates were eliminated, and one article was a retraction of a manuscript. Subsequently, 799 articles were excluded in the initial screening by title and abstract, and 126 articles were included for full-text review. However, two articles could not be obtained, a Polish and a French article published more than 20 years ago. Therefore, 124 articles were evaluated, of which 72 were eligible, 53 for the systematic review since their outcomes had information regarding visual acuity and recurrences, and 39 for meta-analysis since they had similar measures that allowed us to combine the individual results. The articles without information regarding visual acuity or recurrences were not included in the systematic review. More detailed information can be found in Fig 1.

Study characteristics
Of the 72 analyzed studies, 9 were randomized clinical trials, which presented a low risk of bias in the outcomes of interest ( See Fig 2A and 2B). On the other hand, three articles were nonrandomized clinical trials that presented moderate to serious risks of bias for the outcomes (See Fig 3A and 3B). As for the cohort studies, 21 were analyzed and scored low risk of bias (an average of 6.90 out of 8 questions). Eight case-controls were identified and scored low risk of bias (an average of 4.87 out of 5 questions). Regarding the longitudinal studies, four were identified and scored low risk of bias (an average of 1.75 out of 3 questions). Finally, 27 crosssectional articles were identified and scored moderate risk of bias (an average of 5.5 out of 11 questions) [5-7, 9, 11, 12, 14, 25-89].

Clinical and environmental factors
Multiple articles described clinical factors that influence the recurrence rate. One of them was time since the disease onset. Most authors found a higher risk of reactivation in the first years after primary acquired infection, which tends to decrease over time. This risk can reduce by 72% in the first decade, and the decrease is continuous over time [14,44,76,78]. In addition, age over 40 years was a factor that increased the risk of recurrences with a relative risk (RR) up to 1.74, (95% CI 1.06-2.86) [44]. Other clinical factors increasing the risk of OT reactivation were bilateral lesions (RR: 8.0; 95% CI 1.3-50.0) [28], pregnancy (RR: 7.40; 95% CI 4.46-12.06) [33], the presence of a single lesion with a Hazard Ratio (HR) 1.60 (95% CI 1.07-2.40) [12], and an initial active presentation odds ratio (OR) 4.74 (95% CI 1.95-12.91) [27,42].
On the other hand, environmental factors, such as living in regions with high precipitation rates, were also associated with higher recurrence rates. An Argentinian study found that for every mm of precipitation, the frequency of consultations due to OT recurrences increased by 2% in an ophthalmological center (OR: 1.002; 95% CI 1.000-1.003) [54]. Other factor is the consumption of bottled water that reduces recurrences risk [34]; on the other hand, surgeries are controversial as risk factor of reactivation [46,47].
Regarding recurrences in previous and no previous involved eye, they usually occur in the affected eye [7,85]. Finally, the mean number of reactivations was between 0.105 and 2.6 recurrences per year [14,28,31,50]. In addition, reported flare-ups per interval of time were one recurrence in 5 years, 1.7 recurrences in 10 years, and two recurrences in 11 years [42,44,50]. However, it is not possible to meta-analyze this information due to the data heterogeneity.
The development of blindness, as reactivations and visual impairment, was affected by the location of the lesion. It was estimated that central lesions raise 8.95 to 68.6 times the risk of blindness compared to a lesion located outside the macula [7,27,35,37,64]. Additionally, an age �50 years and the presence of lesions size >1 disc diameter (DD) had an OR of 3.27 (95% CI 1. 22-8.8) and 6.30 (95% CI 2. 28-22.46) for blindness, respectively [7,27,64]. Other associated factors are a history of scarring, recurrences during follow-up, and a VA <20/200 at admission [32,35,64]. Interestingly, in one article the geographical region was not statistically significantly associated with reactivations, visual impairment, and blindness [64]. Finally, we

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Ocular Toxoplasmosis Risk factors for recurrences and visual impairment. Europe Crosssectional • Recurrences occurred predominantly in previously affected eyes (with old scars) in contrast to the sporadic cases of recurrence in the healthy contralateral eye (P 0.0001).
• Legal blindness was more common in patients who had ever been treated with corticosteroids without antiparasitic drugs compared to those who received other or no treatments (P = 0.0004) • Legal blindness was more common in patients with a central location of the chorioretinal lesions compared to peripheral lesions (P < 0.0001) • Legal blindness was more common in extensive retinal lesions (>3DD) compared to lesions less than 3DD (P < 0.0001) • Poor visual outcome was more common in congenital toxoplasmosis than in patients with the acute phase of systemic infection (P <0.001) and also in patients with an unknown moment of acquisition of the infection (P = 0.03).
Risk of toxoplasmic retinitis reactivation following intraocular procedures without the use of prophylactic therapy could consider some variables as future biomarkers, such as the evidence of hyporeflective spaces or signal voids in Spectral Domain optical coherence tomography [52]. Nevertheless, in OT patients the standard automated perimetry better indicate the moderate or severe visual impairment (blindness) than visual acuity [51].

Parasite and host factors influencing reactivations and visual acuity outcomes in OT
Factors related to the parasite can influence OT recurrences, L Shobab et al. found that patients infected with serotypes non-reactive (NR) to GRA6 and GRA7 allelic peptide motifs derived  [38].
On the other hand, there are protective and risk factors related to the human host. The AT heterozygosis polymorphisms in the IFN-γ gene at position +874 increases the risk of Genotypes related to low production of IL-10 may be associated with the occurrence of OT. However, it was not related with the recurrences and visual acuity.  Table 3.  recurrence by 49% (HR: 1.49, 95% CI 1.04-2.14) [12]. Additionally, other genetic studies have found that genotypes associated with low production of IL-10 may be associated with the OT

Title Authors (Year) Continent Type of Study Factors related to recurrences in OT Factor that influence visual acuity outcome in OT
Efficacy of specific chemotherapy in the prevention of recurrences of toxoplasmic chorioretinitis during the 4 years following the treatment J.C. Timsit et al. (1987) [5] Europe Nonrandomized controlled study Pyrimethamine-Sulfadiazine therapy appears to be an effective synergistic therapy to prevent recurrent toxoplasmosis uveitis.

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development but not with the recurrence and visual acuity [59]. Likewise, the TNF-α gene polymorphism (2308G/A) do not seem to be associated with OT development and recurrences [58]. Similarly, Ayo et al. found that the presence of the -KIR3DS1/KIR3DL1+/Bw4-8-80Ile + combination was a protective factor against recurrences (OR: 0.13; 95% CI 0.03-0.45) [74]. Finally, a study involving intraocular fluids have found that elevated levels of IL-5 and VEGF positively correlated with recurrences [36]. Also, Silveira C et al. proposed that parasitemia could be a factor related to reactivations, based on the analysis of peripheral blood mononuclear cells (PMBC); however, the sample in this study was small limitating the results generalization [75]. Finally, peptidyl-prolyl cis-trans isomerase A (PPIA) identified by immunoblotting may be a biomarker of multi-episodic disease in OT patients. PPIA can help

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deferring between a first episode of OT, a recurrence of OT, other forms of uveitis, or other parasitic infections during an active ocular inflammation episode [77].

Treatment-related factors influencing reactivations and visual acuity outcomes in OT
Several treatment regimens used in active OT seem to influence the risk of recurrences, such as trimethoprim-sulfamethoxazole (TMP/SMX), spiramycin (Sp), and pyrimethamine sulfadiazine (P+Sdz), in combination with or without systemic and topical steroids. However, studies have not shown that treatment in the disease's active phase can significantly reduce the

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recurrence rate. Prospective studies are needed to analyze this outcome in more detail [5,9,31,45,61,71]. However, some studies debate this; proposing the use of antibiotic therapy is not superior to the use of no management at all during active episodes of OT [90,91]. This issue still needs more studies with optimal methodological quality [31]. Additionally, the use of systemic steroids without antibiotic treatment and subconjunctival steroids are a determining factors for the presence of new recurrences [42].
On the other hand, studies have shown that the regimen of azithromycin alone or with a steroid has less effectiveness than other schemes [61,71], achieving comparable effectiveness

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only when combined with pyrimethamine [45]. However, some of these studies have a poor methodological quality, so their conclusions do not have a high validity. Therefore, the information regarding the use of azithromycin for recurrences should be analyze cautiously (see Fig 3A and 3B and S2 Table).
The use of pyrimethamine/sulfadoxine (Sdx) as secondary antifolate prophylaxis can lead to 3,5-year recurrence-free survival in 90.9% of cases (Scheme: P+Sdx 25 mg/500 mg, one tablet twice a week for six months without folinic acid supplementation) [11]. Another proposed scheme of prophylaxis is the single tablet TMP160 mg/SMX 800 mg every three days for up to 20 months, showing a 75% reduction of recurrences with a HR of 0.25 (95% CI, 0.08-0.75) [53]. Also, another scheme with TMP/SMX was described by Fernandes Felix et al. where they gave one dose of TMP160 mg/SMZ 800 every other day for 311 days, showing a single recurrence in 6 years in the experimental group [9]. Moreover, two studies report the use of photocoagulation for the prevention of reactivation; however, these studies do not have much methodological soundness and contradict each other [60,81]. Additionally, in immunosuppressed patients, the prophylactic scheme with P+Sdz seems to reduce the rate of recurrences, however, more studies are needed [84].
Regarding VA, a significant effect of antibiotic therapy is observed in general, but more studies of better quality are needed to make any affirmation [43,45,72,87,89].
Prophylaxis treatment to reduce recurrence. This analysis sought to determine the effect of prophylactic treatments reducing recurrences [9,53]. Fig 9A, shows the effect at the first year after the TMP/SMX prophylaxis vs placebo OR 0.17 (95% CI; 0.03-0.86; I2 = 31% (P = 0.03)). Fig 9B shows the effect at the second year after the TMP/SMX prophylaxis vs placebo, 0.13 (95% CI; 0.02-0.81; I2 = 43% (P = 0.03)). In this case, a statistical significant result was evidenced, also, a tendency of reduction in the risk of recurrence was incremental over time.

Discussion
Our systematic review and meta-analysis evaluated the effect of multiple clinical, environmental, geographic, parasite, genetic, and management factors on the recurrences, visual impairment, and blindness in OT.
One of the most critical factors related to recurrences was the first year after the first lesion because the risk tends to decrease in the subsequent years [7,14]. Additionally, age >40 years, binocularity of the lesions, pregnancy, precipitation, parasite with a non-reactive serotype, AT heterozygosis in the IFN-γ gene at position +874, and the use of steroids without antibiotics as

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treatment were the main determinants of reactivations. The majority of these factors can be explained by the the immunosenescence that allows reactivations of the parasite cyst located in the retina [12,14,27,44]. Therefore, these are the variables that should be considered when elaborating additional recommendations [14,28,38,42,44,54]. Additionally, we meta-analyzed the prevalence of recurrences, visual impairment, and blindness worldwide. We found that recurrences are most common in South American and Central American patients, corroborating that these latitudes have more severe cases of OT, possibly due to more virulent strains [92,93]. However, all studies in Central America evaluated only one region (Cuba), which can generate an overestimation of the effect. On the other hand, it should be considered that the meta-analysis of prevalence of recurrence has a high risk of publication bias, which is why they should be analyzed carefully, and the subgroup's analysis did not entirely correct them. Owing to this, we suggest that there are additional factors that explain the high heterogeneity. For example, the serotype could be another factor, as L Shobab et al. [38] reported that the NR serotype had the highest risk of recurrence, which may be considered in future studies.
Regarding visual acuity and blindness prevalence in OT eyes, we found that in South America there is a greater prevalence of visual impairment and blindness than in Europe but not in the rest of the world. However, some studies' incorrect reports of this outcome can limit our results. Additionally, it should be noted that the sub-analysis by subgroups appropriately controlled the heterogeneity of the metanalysis.
In the analysis of risk factors associated with blindness, we found that recurrences and the macular localization of the lesion are the most important factors related to blindness. This has been supported by previous studies, which evidenced that macular lesions have up to 8.95 to 68.6 times increased risk of blindness [7,27,35,37,64]. Therefore, as clinicians, we have to prevent these scenarios to reduce the visual impact of OT.
In addition to the previously mentioned factors (macular location of the lesions and recurrences) [7,27,32,35,37,64,65], other factors that increased the risk of blindness are extensive or atypical lesions in patients aged more than 45 years [7,27,32,64,79], and congenital toxoplasmosis [7,57,66]. Therefore, a closer follow-up and optimized treatment should be considered in these patients. Moreover, we evidence that few articles detail the immunological status of patients, which should be added in future studies in order to quantify the impact of this variable. Regarding the treatment in active lessons, we found that TMP/SMX and clindamycin (intravitreal and subconjunctival) are possibly superior to PS in preventing recurrence; nevertheless, these differences were not statiscally significant and must be interpreted with caution. Prospective studies are needed to evaluate the best therapy in the acute phase of the disease that further prevents recurrences.. Although there is a lack of data, current evidence can be useful to guide prophylactic treatment [39,43,56,62,67].
Prophylaxis in patients with OT is a protective factor that increases over time, reaching a reduction of almost 87% compared to placebo. This metanalysis does not have a high risk of publication bias. Also, it is essential to consider there were evaluated few studies; we suggest conducting more studies in this field to expand the evidence and assess the superiority of the different schemes. Our results are supported by the marked reduction trend in recurrences that has been reported with the use of antibiotic prophylaxis with TMP/SMX in any scheme, more detailed information on schemes, doses and times see Table 4 [9,53]. Nevertheless, a publication by Silveira et al. showed after discontinuing the TMP/SMX treatment, the recurrence rate is similar to the placebo group, even after ten years of use. This is explained due to a loss of the drug's effect once discontinued [8]. This differs from Fernandes Felix et al., who have shown a much lower recurrence rate after the discontinuation of the prophylactic scheme, with six years free of recurrences [9]. We proposed that other possible variables could be involved, such as the periodicity of the prophylactic treatment, parasite genotype, genetic susceptibility of the host, and the immune status, that may differ from the studies' populations [9,11,53]. We suggest performing further studies on these additional variables.
In conclusion, considering the results of our meta-analysis and the evidence previously presented, we suggest that patients older than 40 years, patients with de novo OT lesions or with less than one year after the first episode, macular area involvement, lesions greater than 1DD, congenital toxoplasmosis, and bilateral compromise could benefit from using prophylactic therapy, especially if they live in South America or in a high precipitation area. However, due to the number of studies and their characteristics, it is not possible to determine which prophylactic strategy is superior [9,11,53].